Cytochromes P450 (CYP) are the principal enzymes for the oxidative metabolism of many drugs, procarcinogens, promutagens, and environmental pollutants. Cytochrome P450 is a heme-containing, membrane-bound, multienzyme system that is present in many tissues in vivo but is present at the highest level in liver. In human liver, it is estimated that there are 15-20 different xenobiotic-metabolizing cytochrome P450 forms. A standard nomenclature based on relatedness of amino acid sequences has been developed. Certain P450 forms (such as CYP3A4 and CYP2C19) are known to be polymorphic in humans and some (such as CYP1A2 and CYP3A4) are regulated in response to environmental chemicals. Competition for metabolism by a particular cytochrome P450 form is a principal mechanism of some clinically significant drug-drug interactions.
Identification of the enzymes responsible for metabolism is becoming an important aspect of drug development. Such identifications consider both the metabolism of the new drug as well as inhibition by the new drug. The identification of enzymes involved in metabolism of the new drug allows prediction, based on knowledge of the ability of coadministered drugs to inhibit the same enzymes, of which coadministered drugs may inhibit the metabolism of the new drug. This information can also be used to predict individual variability based on known metabolic polymorphisms. The identification of the enzymes most sensitive to inhibition by the new drug allows prediction, based on knowledge of which coadministered drugs are metabolized by the same enzyme, of which coadministered drug's metabolism may be inhibited by the new drug. Obtaining information for a series of drug candidates early in the drug discovery process can assist in the choice of the best drug candidate for further development.
CYP3A4 is the most abundant cytochrome P450 in the human liver and intestine. CYP3A4 is responsible for the metabolism of many important drugs, for example: opioid analgesics, corticosteroids, immunosuppressants, and antiarrhythmics (Rendic, S. and F. J. Di Carlo (1997) Drug Metab Rev. 29, p.413-580). Inhibition of this enzyme by dietary compounds (for example, grapefruit juice) or drugs has been responsible for several clinical drug-drug interactions. The importance of this P450 in drug metabolism makes the screening for metabolism and inhibition of this enzyme important in drug development.
Assays for CYP3A have focused on the metabolism of drug molecules or drug candidates. Substrates such as testosterone or midazolam are effective in assessing CYP3A activity and inhibition, but are not amenable to high throughput screening assay technology (both require time consuming separation of CYP3A reaction products using HPLC). Also, neither of these substrates have the necessary fluorescent properties that make the substrate useful for in situ fluorescent plate analysis.
We have previously reported the use of the commercially available compound 7-benzyloxyresorufin (BzRes) as a fluorescent substrate for assessing CYP3A4 activity in a high throughput mode (See Crespi et al. Anal Biochem. 248, 188-190, (1997). However, the low enzymatic turnover and poor specificity of this substrate make it of limited utility.
The O-dealkylation of 7-alkoxycoumarins has been reported as a fluorometric assay for determining the metabolic differences between cytochrome P450 isoforms using microsomes from several rat tissues (Kobayashi, Y., Fang, X, Szklarz, G. D., and J. R. Halpert, (1998) Biochem. 37, pp6679-6688). The O-dealkylation of 7-pentoxy-, 7-hexoxy-, and 7-benzyloxycoumarin have been studied in rat liver microsomes (Mayer, R. T., Netter, K. J., Heubel, F., Hanemann, B, Buchheister, A., Mayer, G. K. and M. D. Burke, (1990) Biochem. Pharmacol. 40, pp1645-1655). 7-Ethoxy-4-trifluoromethylcoumarin is O-dealkylated by human CYP2B6 and other human P450s (Code et al., (1997) Drug Metab. Dispo. 25, pp985-993; Morse, M. A., and J. Lu (1998) J. Chrom. B, 708, pp290-293.). The first coumarin analog whose O-dealkylation is specific for a single human P450 was recently developed by us (U.S. Ser. No. 60/092,995, entitled Novel CYP2D Fluorescent Assay Reagents).